Project Summary. Osteoarthritis (OA) is the most common arthritic condition in the U.S.A., and is responsible in aggregate for vast economic costs, due both to direct medical care and to lost work days. OA is primarily a degenerative process of articular cartilage and our research focus is to identify strategies to stimulate cartilage matrix repair. In cartilage, cell-matrix interactions are the primary means for chondrocytes to sense changes in the extracellular environment, and signal a reparative response. Hyaluronan (HA) binding to the CD44 receptor is a key player in such interactions. Since HA-CD44 interactions are required for the retention of proteoglycan in the matrix, the relationship between these components is crucial to cartilage homeostasis. For chondrocytes, CD44 signaling is initiated by disruption of HA-CD44 interactions and thus declustering of CD44. Loss of HA- CD44 interactions exerts a negative effect on the canonical BMP/BMP-R/Smad1 signaling pathway that can be rescued by the addition of HA. Furthermore, changes in HA-CD44 interactions initiated other signaling cascades resulting in the stimulation of genes for matrix turnover (MMP-3, MMP-13, iNOS) and for matrix repair (collagen II, aggrecan, HAS2, BMP7). As such this exciting discovery defines a new chondrocyte-based model that closely mimics the early stage of OA namely, attempted repair but repair that is coincident with enhanced matrix degradation. The first aim will define the signaling events initiated by the loss of cell-matrix interactions in chondrocytes that give rise to stimulation of both matrix repair and matrix degradation. Both loss-of-function and augmentation-of-function approaches with murine, bovine and human chondrocytes will be taken to discern the role of CD44 in the responses. In the second aim, the chondrocyte dedifferentiation/re- differentiation model will be used. Dedifferentiated articular chondrocytes exhibit changes in phenotype, some of which mimic osteoarthritic chondrocytes, importantly a reduced capacity for repair. Dedifferentiated chondrocytes exhibit a naturally-occurring degradation (shedding) of the CD44 extracellular domain as well as a secretase-mediated generation of the CD44 intracellular domain (ICD) which could initiate CD44 declustering. Nevertheless, these cells are capable of re-differentiation to a more chondrocyte-like phenotype. The mechanism for this reversion is related to changes in the matrix, the cytoskeleton and in intracellular signaling - all events that could be mediated by CD44 and will be examined in our studies. Accordantly, chondrocytes from OA patient also exhibit CD44 fragmentation and the potential for re-differentiation of human OA cells will also be examined. In specific aim 3, our hypothesis that HA-CD44 interactions enhance cartilage repair will be examined in studies on the formation of ectopic cartilage in vivo in CD44 knockout and wildtype mice and the generation of neo-cartilage disks in vitro. Together these aims will contribute to our understanding of HA-CD44 interactions in the regulation of chondrocyte metabolism.